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* Department of Dermatology, and
Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06520
Ag processing and presentation via MHC class II is essential for activation of CD4+ T lymphocytes. 
-IFN-inducible lysosomal thiol reductase (GILT) is present in the MHC class II loading compartment and has been shown to facilitate class II Ag processing and recall responses to Ags containing disulfide bonds such as hen egg lysozyme (HEL). Reduction of proteins within the MHC class II loading compartment is hypothesized to expose residues for class II binding and protease trimming. In vitro analysis has shown that the active site of GILT involves Cys46 and Cys49, present in a CXXC motif that shares similarity with the thioredoxin family. To define the functional requirements for GILT in MHC class II Ag processing, a GILT-deficient murine B cell lymphoma line was generated and stably transduced with wild-type and cysteine mutants of GILT. Intracellular flow cytometric, immunoblotting, and immunofluorescence analyses demonstrated that wild-type and mutant GILT were expressed and maintained lysosomal localization. Transduction with wild-type GILT reconstituted MHC class II processing of a GILT-dependent HEL epitope. Mutation of either Cys46 or Cys49 abrogated MHC class II processing of a GILT-dependent HEL epitope. In addition, biochemical analysis of these mutants suggested that the active site facilitates processing of precursor GILT to the mature form. Precursor forms of GILT-bearing mutations in Cys200 or Cys211, previously found to display thiol reductase activity in vitro, could not mediate Ag processing. These studies demonstrate that the thiol reductase activity of GILT is its essential function in MHC class II-restricted Ag processing.
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1 This study was supported by the Howard Hughes Medical Institute (to P.C.), the Dermatology Foundation, and the Natural Sciences and Engineering Research Council of Canada and Le Fonds québécois de recherche sur la nature et les technologies (to R.L.L.). K.T.H. is the recipient of the Dermatologist Investigator Research Fellowship.
2 Current address: Department of Basic Medical Sciences, University of Arizona College of Medicine, Phoenix, AZ 85004.
3 Address correspondence and reprint requests to Dr. Peter Cresswell, Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, P.O. Box 208011, New Haven, CT 06520-8011. E-mail address: peter.cresswell{at}yale.edu
4 Abbreviations used in this paper: Ii, invariant chain; CLIP, class II-associated Ii peptide; GILT, -IFN-inducible lysosomal thiol; HEL, hen egg lysozyme; MSCV, murine stem cell virus.
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